circ_0007928/HuR/HIF1-α 轴调控肺腺癌侵袭转移的机制研究

By analyzing our previous microarray data, we identified circ_0007928 ( CI ) as a low expressed novel circRNA in human lung adenocarcinoma. We validated the expression level of CI by qRT-PCR and found that it is almost 4.28-fold down-regulated in cancer tissues when compared with adjacent normal lung tissues. The expression level of CI is negatively correlated with the lymph node metastasis and positively correlate with the long –term survival time of patients significantly. In vitro experiment indicated that over-expression of CI repressed the invasion and metastasis of lung adenocarcinoma cell lines. Bioinformatic analysis and RIP experiment revealed that CI could interact with HuR, an RNA binding protein, to decrease mRNA stability of HIF1-α, a known target gene of HuR. Based on the above clinical and experimental data, we hyppthesized that “ CI could decoy HuR to decrease HIF1-α expression at post-transcription level, which inhibited the invasion and metastasis of lung adenocarcinoma”. The current project aims to assess biological function of CI to repress the invasion and metastasis. Secondly, the mechanism of circ_0007928/HuR/HIF1-α axis to regulate the metastasis in lung adenocarcinoma will be deeply validated by RNA pull down, FISH and rescue experiments. Finally the mechanism will be confirmed in animal experiment and clinical lung adenocarcinoma patients. This project may provide the new way for the prevention and treatment of lung adenocarcinoma.

circ_0007928（简称CI）是申请人前期通过筛选获得的一条低表达于肺腺癌环状RNA。前期验证显示CI在肺腺癌表达下调4.28倍，与淋巴结转移负相关，表达越低预后越差；进一步体外实验表明过表达CI抑制肺腺癌细胞株侵袭及转移。生物信息学预测及RIP等实验表明CI可与RNA结合蛋白HuR相结合，降低HuR下游促转移基因HIF1-α的mRNA稳定性。基于上述临床与基础的证据，本项目提出“CI 通过诱捕HuR蛋白，在转录后水平下调HIF1-α 的表达，从而抑制肺腺癌侵袭转移”的科学假说。本研究拟运用体内外实验进一步验证CI抑制肺腺癌侵袭转移生物学功能；采用RNA pull-down、FISH-荧光共定位及交叉拯救等实验阐明CI/HuR/HIF1-α轴调控肺腺癌侵袭转移的分子机制；并在动物及临床水平进一步验证该分子机制。本研究可能为肺腺癌防治提供新的思路。

环状RNA（circRNAs）是一类共价单链环形的非编码RNA，其在真核细胞内稳定表达。随着RNA测序和生物信息学的进步，大量的circRNA被鉴定参与多种生理和病理学进程，尤其是在肿瘤的发生发展过程。肺癌（Lung Cancer）是世界上发病率和死亡率最高的恶性肿瘤，其中肺腺癌（LUAD）是最常见的病理亚型。尽管随着新辅助化疗及免疫治疗等方法的应用，肺腺癌的治疗预后有了一定的提升，但其5年生存率仍然低于15%。其中最主要的危险因素就是肿瘤的复发转移。.HuR蛋白是经典的癌症相关RNA结合蛋白（RBP）。HuR蛋白可以结合线性RNA的3’UTR区以促进线性RNA的稳定。有报道指出许多circRNA可以与HuR蛋白相结合。但circRNA与HuR结合在肺腺癌中的机制仍然不清楚。.课题组综合分析A549中circRNA的表达谱及HuR的CLIP数据，筛选出来源于DCUN1D4基因2-6外显子的circRNA-circDCUN1D4。肺腺癌中，高表达的DHX9显著抑制circDCUN1D4的成环，使其显著低表达。功能实验证实在体内外，CircDCUN1D4均显著抑制了肺腺癌细胞的的侵袭转移能力。机制研究上，课题组发现circDCUN1D4在细胞内与HuR蛋白结合并促进HuR蛋白的胞质转运。同时circDCUN1D4在胞质内可以作为一个“脚手架”，不仅可以与HIF1a的mRNA相结合，同时也可以通过与TXNIP mRNA 3’UTR的互补引导HuR蛋白与TXNIP mRNA的结合，以稳定TXNIP mRNA，促进TXNIP的表达。临床上，circDCUN1D4的表达水平与肺腺癌患者的淋巴结转移情况及预后显著负相关。本研究证实circDCUN1D4通过与HuR蛋白及TXNIP mRNA共同形成三元复合物以抑制肺腺癌的转移及糖酵解能力。